In vitro cell culture is routinely performed as part of a wide variety of biological research and development programs. In their most common form, cell based experiments are carried out in culture dishes or flasks under static conditions—i.e., those in which no external forces are applied to the cells. Work conducted with statically grown cells has led to many breakthroughs in fields such as cell biology, biochemistry, immunology, and cancer research. However, the inability of static culture to accurately mimic the behavior of cells in dynamic tissue environments constitutes a boundary on the usefulness of this technique. This is illustrated by the number of drug candidates that fail at the transition from in vitro to in vivo testing. It is well known that environmental forces (such as those derived from the flow of blood and other interstitial fluids) influence the behavior of cells and tissues in determining states of health and disease and responses to biochemicals (Buchanan et al., 1999; Urbich et al., 2001; Wasserman & Topper, 2004; Sheikh et al., 2005; Chatzizisis et al., 2007; Chiu et al., 2007; Tsai et al., 2007). Similarly, these forces can also modulate cellular responses to pharmaceuticals, and influence their ultimate efficacy profiles. Since static culture is incapable of introducing variables such as fluid flow into experimental design, alternative means of cell cultivation are necessary to investigate the influence of physiological forces on cell behavior, both in native environments and in response to biochemicals and pharmaceuticals.
The effects of blood flow on cell physiology were first observed in the context of arterial cells susceptible to developing arterial (heart) disease, but other physiological phenomena, such as immune cell recruitment, wound-healing, stem cell differentiation, and tissue regeneration are also known to be force dependent (Rinker et al., 2001; Dekker et al., 2002; Burns & DePaola, 2005; LaMack et al., 2005; Yamamoto et al., 2005; McKinney et al., 2006). Due to the prevalence of heart disease in western society, the effect of fluid flow has become a primary topic of investigation for those interested in understanding its pathology and developing novel treatments. Due to the dependence of the development and progression of heart disease on the characteristics of arterial blood flow, much research is focused upon understanding how various fluid forces influence cell physiology. This work cannot be performed under static conditions, but instead requires the use of dynamic culture systems. Similarly, investigations into the other force dependent physiological processes mentioned above have related culture system requirements. Unfortunately, there has been no commercially available consumable device flexible enough to support the variety of fluid force based cell culture research and development that is being conducted. Instead, most academic and commercial laboratories have created their own systems, while a large number of other entities that would like to perform such experiments do not, as they consider the need to fabricate and assemble the required apparatus as a significant barrier to practice.
In addition to the areas of research and development currently investigated in flow systems, there is a need to expand this approach to the drug discovery pipeline. The same blood vessel cells involved in heart disease serve as gatekeepers for drugs entering the bloodstream, and participate in determining their efficacy (McNeish, 2004; LaMack et al., 2005). Kidney tubular epithelial cells and liver sinusoidal epithelial cells are involved in drug metabolism and excretion, are subject to fluid flow, and their flow sensitivity has been reported (Duan et al., 2010; Essig and Friedlander, 2003; Shah et al., 1997). By conducting initial screening experiments and later toxicity/therapeutic studies with cell cultures exposed to conditions similar to those that exist within the body, results will be more closely linked to actual behavior in tissue, and the economics of the process improved. It is our belief that this can only be achieved through the use of a device such as the chamber device proposed in this application. These outcomes will allow pharmaceutical companies to identify high value candidates earlier, to understand their properties more completely, and to focus their resources on only those molecules that meet the more realistic set of physiological criteria.
There are two common types of devices that support cell and tissue experiments in a dynamic fluid environment. The first of these is the parallel plate flow chamber. Parallel plate flow chambers consist of two parallel plates separated by a gap that forms the flow channel. This gap is generally created by a gasket or spacer that is used to simultaneously seal the flow channel and separate the plates. Fluid is introduced from one end of the chamber and exits on the one opposite. Parallel plate devices are commonly used for exposing cells to defined levels of shear stress, applying specific flow characteristics, and for investigating cell to cell or cell to substrate attachment properties (Frangos et al., 1985; Rinker et al., 2001; McKinney et al., 2006; Shepherd et al., 2009; Shepherd et al., 2011). The other type of device consists of a cone and plate viscometer that has been modified to support cell cultures. In these systems, cells may be exposed to various levels of fluid shear stress and flow waveforms created by the rotation of the cone (Dai et al., 2004). Neither of these systems are currently commercially available for large scale culture activities. Some flow chambers based on a parallel plate design are being marketed by companies such as Ibidi, Fluxion, Cellix, Cellasics, Integrated Biodiagnostics, and Glycotech; however most are based upon small microfluidic flow channels, and do not provide for a wide variety of flow conditions or readout modalities. Additionally, some chambers have issues generating uniform flow (and hence shear stress) distribution (Nauman et al., 1999; Brown & Larson, 2001; McCann et al., 2005; Anderson et al., 2006).
Described herein are flow chambers with differing geometries, obstacles, gap widths, wall heights, etc. designed to provide finely tunable flow conditions, as well as methods of making and using the same to assay various biological properties of cells and/or tissues experiencing different flow conditions.